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Abstract:

The invention relates to modulation of fungal morphology between
yeast-to-hyphal growth transition by controlling muramyl-L-alanine
concentration and uses thereof.

Claims:

1. A method of modulating morphogenesis of a fungus by controlling the
concentration of muramyl-L-alanine in the fungal environment.

2. The method as claimed in claim 1 wherein the fungal environment is in a
body fluids.

3. The method as claimed in claim 1 wherein the fungal environment is in
serum.

4. The method as claimed in claim 1 wherein the fungus is a yeast.

5. The method as claimed in claim 4 wherein the yeast is a Candida.

6. The method as claimed in claim 5 wherein the Candida is Candida
albicans.

7. The method as claimed in claim 1 wherein increasing the concentration
of muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
the core structure induce the fungus to a hyphal morphogenesis.

8. The method as claimed in claim 1 wherein removing, degrading or
neutralizing the concentration of muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in the core structure inhibits the fungus to a
hyphal morphogenesis.

9. The method of claim 8 wherein muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in the core structure is removed, degraded or
neutralised by a muramyl-L-alanine-specific antibody.

10. The method of claim 9 wherein the antibody is catalytic.

11. A method for treating a patient to at least reduce Candida hyphal
growth, which comprises the step of contacting the infection with an
antagonist to muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in the core structure.

12. The method of claim 11 wherein the antagonist is an antibody to
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in the
core structure.

13. The method of claim 12 wherein the antibody is a neutralizing antibody
to muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
the core structure.

15. The method of claim 11 wherein the antagonist comprises a compound
with a muramyl-L-alanine in the core structure.

16. The method of claim 15 wherein the compound engages the LRR domain of
CaCdc35p preventing muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in the core structure from binding to the LRR domain.

17. A method of modulating morphogenesis of a fungus in a patient
comprising the steps of:a. preparing a composition comprising an adjuvant
and a compound with a muramyl-L-alanine in the core structure; andb.
administering the composition to the patient.

18. A composition comprising a therapeutically effective amount of a
modulator of muramyl-L-alanine concentration.

19. The composition of claim 18 wherein the modulator is an antagonist to
muramyl-L-alanine.

20. The composition of claim 19 wherein the antagonist is an antibody to
muramyl-L-alanine.

21. The composition of claim 20 wherein the antibody is a catalytic
antibody to muramyl-L-alanine.

22. The composition of claim 18 wherein the modulator comprises a compound
with a muramyl-L-alanine in the core structure.

23. The composition of claim 22 wherein the compound engages the LRR
domain of CaCdc35p preventing muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in the core structure from binding to the LRR
domain.

24. The composition of claim 22 further comprising an adjuvant capable of
inducing an immune response.

25. (canceled)

26. (canceled)

27. A method of determining the extent of Candida hyphal growth in a
sample comprising the step of measuring the amount of muramyl-L-alanine
and/or compounds that include muramyl-L-alanine in their core structure
in body fluids sampled from a Candida infection.

28. The method of claim 27 further comprising the step of determining the
patient's treatment according to whether hyphal growth is expected or
not.

29. A method for screening for agonists and antagonists of Candida hyphal
growth comprising the steps of:a. contacting (i) muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure and (ii)
the LRR domain of CaCdc35p from a Candida species with a sample compound;
andb. detecting whether the sample compound exhibits agonistic or
antagonistic activity towards the interaction.

30. The method of claim 29 wherein the antagonistic drugs are capable of
interfering either in a direct or indirect manner with the interaction
between Candida and muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure.

31. (canceled)

32. A method for treating a patient infected with Candida comprising
administering to the patient the composition of claim 18.

33. A method for treating a patient infected with Candida comprising
administering to the patient an antagonist of Candida hyphal growth,
wherein the antagonist is identified according to the method of claim 29.

Description:

FIELD OF THE INVENTION

[0001]The invention relates to modulation of fungal morphology between
yeast-to-hyphal growth transition and use thereof.

[0004]Feng et al. (1999) first found that the majority of the serum hyphal
inducer(s) can pass through a dialysis membrane with a molecular weight
cut-off of 1 kDa (Feng, Q. et al (1999). J. Bacterial. 181, 6339-6346.).
Hudson et al. (2004) recently reported that there are two distinct hyphal
inducers in serum (Hudson, D. A., et al (2004) Microbiology 150,
3041-3049). Glucose was described to be a dialyzable inducer responsible
for -80% of the inducing activity. A minor inducer was found to be
non-dialyzable and trichloroacetic acid-precipitable. This report
observed that adding the dialyzable fraction to glucose-containing medium
did not induce the yeast-hypha switch.

[0007]The present invention seeks to provide a hitherto unknown means for
modulating hyphal growth. It provides methods for screening modulators
that are capable of achieving this outcome as well as therapeutically
active compounds that are able to interfere with C. albicans virulence.

General

[0008]Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications other
than those specifically described. The invention includes all such
variation and modifications. The invention also includes all of the
steps, features, formulations and compounds referred to or indicated in
the specification, individually or collectively and any and all
combinations or any two or more of the steps or features.

[0009]Each document, reference, patent application or patent cited in this
text is expressly incorporated herein in their entirety by reference,
which means that it should be read and considered by the reader as part
of this text. That the document, reference, patent application or patent
cited in this text is not repeated in this text is merely for reasons of
conciseness.

[0010]Any manufacturer's instructions, descriptions, product
specifications, and product sheets for any products mentioned herein or
in any document incorporated by reference herein, are hereby incorporated
herein by reference, and may be employed in the practice of the
invention.

[0011]The present invention is not to be limited in scope by any of the
specific embodiments described herein. These embodiments are intended for
the purpose of exemplification only. Functionally equivalent products,
formulations and methods are clearly within the scope of the invention as
described herein.

[0012]The invention described herein may include one or more range of
values (e.g. size, concentration etc). A range of values will be
understood to include all values within the range, including the values
defining the range, and values adjacent to the range which lead to the
same or substantially the same outcome as the values immediately adjacent
to that value which defines the boundary to the range.

[0013]Throughout this specification, unless the context requires
otherwise, the word "comprise" or variations such as "comprises" or
"comprising", will be understood to imply the inclusion of a stated
integer or group of integers but not the exclusion of any other integer
or group of integers. It is also noted that in this disclosure and
particularly in the claims and/or paragraphs, terms such as "comprises",
"comprised", "comprising" and the like can have the meaning attributed to
it in U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting essentially
of" and "consists essentially of" have the meaning ascribed to them in
U.S. Patent law, e.g., they allow for elements not explicitly recited,
but exclude elements that are found in the prior art or that affect a
basic or novel characteristic of the invention.

[0014]Other definitions for selected terms used herein may be found within
the detailed description of the invention and apply throughout. Unless
otherwise defined, all other scientific and technical terms used herein
have the same meaning as commonly understood to one of ordinary skill in
the art to which the invention belongs.

SUMMARY OF THE INVENTION

[0015]The present invention is derived from the discovery that
muramyl-L-alanine and compounds that include muramyl-L-alanine in their
core structure such as bacterial peptidoglycan compounds like
muramyl-L-alanine, muramyl-L-alanyl-D-isoglutamine,
N-acetyl-muramyl-L-alanine, and N-acetyl-muramyl-L-alanyl-D-isoglutamine,
constitute the principal Candida hyphal inducers in body fluids. These
compounds bind with specific affinity within the leucine-rich-repeats
(LRR) domain of CaCdc35p; an essential upstream regulator for hyphal
growth in Candida (e.g. Candida albicans). Furthermore, LRR domain
mutations induced in CaCdc35p abolished (a) the binding of
muramyl-L-alanine and compounds that include muramyl-L-alanine in their
core structure, and (b) hyphal growth.

[0016]Thus, the invention provides a method for treating a patient to at
least affect Candida hyphal growth, which comprises the step of:
contacting the infection with (a) an antagonist to muramyl-L-alanine
and/or compounds that include muramyl-L-alanine in their core structure
and/or (b) a compound that engages the LRR domain of CaCdc35p preventing
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure from binding to the LRR domain. Preferably, the
antagonist interferes with Candida hyphal growth by means that remove,
degrade, neutralize or compete with muramyl-dipeptide related compounds
in a patient's body fluids (such as without limitation, blood, plasma,
bodily fluids in esophagus, throat, interstitial fluid, lymph, mucus,
etc). More preferably, the antagonist is effective against
muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanine,
N-acetylmuramyl-L-alanyl-D-isoglutamine or
muramyl-L-alanyl-L-isoglutamine. In a highly preferred form, the
antagonist is specifically effective against
muramyl-L-alanyl-D-isoglutamine.

[0017]An alternative form of the present invention resides in
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure in the manufacture of a medicament for treating a
patient infected with Candida, preferably a medicament used in treatment
to affect candida hyphal growth.

[0018]The present invention also relates to compositions including
pharmaceutical compositions comprising a therapeutically effective amount
of (a) an antagonist to muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure and/or (b) a compound that
engages the LRR domain of CaCdc35p preventing muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure from
binding to the LRR domain. As used herein a compound will be
therapeutically effective if it is able to affect Candida hyphal growth.
The compound may further comprise an adjuvant capable of inducing an
immune response in a patient.

[0019]The invention also provides a means for prognosing or diagnosing the
course of a Candida infection, comprising the steps of: measuring the
amount of muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure in body fluids sampled from a
Candida infection.

[0020]Further the invention provides a means for determining the most
effective treatment for a Candida infection, comprising the steps of:
diagnosing the state of hyphal growth according to the above method and
then determining the patient's treatment according to whether hyphal
growth is expected or not.

[0021]Consistent with the invention there is provided a means for
screening for agonists and antagonists of Candida hyphal growth
comprising the steps of: (a) contacting (i) muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure and (ii)
the LRR domain of CaCdc35p from a Candida species with a sample compound,
and (b) detecting whether the sample compound exhibits agonistic or
antagonistic activity towards the interaction. Preferably, the method is
used to screen for antagonistic drugs that are capable of interfering
either in a direct or indirect manner with the interaction between C.
albicans (such as, muramyl-L-alanine, muramyl-L-alanyl-D-isoglutamine,
N-acetyl-muramyl-L-alanine, N-acetyl-muramyl-L-alanyl-D-isoglutamine,
muramyl-L-alanyl-L-isoglutamine).

[0022]The present invention also relates to compounds identified by the
above method and their use in treating Candida hyphal growth in a
patient.

[0023]In another aspect of the invention a method of modulating
morphogenesis of a fungus by controlling the concentration of
muramyl-L-alanine in the fungal environment. The fungal environment may
include body fluids (such as without limitation, blood, plasma, bodily
fluids in esophagus, throat, interstitial fluid, lymph, mucus, etc). The
fungus may be a yeast such as Candida or Candida albicans. The modulation
may include inducing hyphal morphogenesis by increasing the concentration
of muramyl-L-alanine or inhibit the hyphal morphogenesis by removing
degrading or neutralizing the concentration of muramyl-L-alanine. The
concentration of muramyl-L-alanine may be removed degraded or neutralised
by an antibody such as a catalytic antibody.

[0024]Accordingly, the methods described herein may be used in prognostic,
diagnostic, therapeutic and drug screening methods. Other aspects and
advantages of the invention will become apparent to those skilled in the
art from a review of the ensuing description, which proceeds with
reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1: Chemical structures of hyphen a muramyl-L-alanine that does
not have the N-acetyl group commonly found in bacterial PGNs and
muramyl-L-alanyl-D-isoglutamine.

[0026]FIG. 2: Shows that 2% of the filtrate of TFA treated serum induced
more than 90% of the yeast cells to switch to hyphal growth.

[0029]FIG. 5: Solid phase synthesis of hyphin a muramyl-L-alanine that
does not have the N-acetyl group commonly found in bacterial PGNs

DETAILED DISCLOSURE OF THE INVENTION

[0030]The present invention derives from the applicant's discovery that
muramyl-L-alanyl-D-isoglutamine is approximately 300 times more active
than N-acetylglucosamine as an inducer of Candida hyphal growth.
N-acetylglucosamine is currently the most potent single-compound hyphal
inducer known. Further, this research has also revealed that
muramyl-L-alanine-D-isoglutamine binds with high affinity within the LRR
domain of CaCdc35p and that LRR-domain mutations induced in CaCdc35p
abolished muramyl-L-alanyl-D-isoglutamine binding and hyphal growth.
These data suggest that CaCdc35p plays a role in signal recognition as
well as signal transduction. These data reveal that CaCdc35p plays
central a role in signal recognition as well as signal transduction.
These findings unveil an important human pathogenic factor for Candida
infection and the use of an evolutionarily conserved mechanism in this
pathogen-host interaction.

Method for Treating a Patient with a Candida Infection

[0031]On the basis of the above, the present invention provides a method
for treating a patient with a Candida infection or an infection from a
related organism, which comprises the step of: contacting the infection
with (a) an antagonist to muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure and/or (b) a compound that
engages the LRR domain of CaCdc35p preventing muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure from
binding to the LRR domain. Desirably, the antagonist is provided in a
therapeutic effective amount.

[0032]An alternative form of the present invention resides in
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure in the manufacture of a medicament for treating a
patient infected with Candida, preferably a medicament used in treatment
to affect candida hyphal growth.

[0033]"Treatment" and "treat" and synonyms thereof refer to both
therapeutic treatment and prophylactic or preventative measures, wherein
the object is to prevent or slow down (lessen) a Candida condition, in
particular Candida Hyphal growth. Those in need of such treatment include
those already with a Candida infection as well as those prone to getting
it or those in whom a Candida infection is to be prevented.

[0034]As used herein a "therapeutically effective amount" of a compound
will be an amount of active agent that is capable of preventing or at
least slowing down (lessening) a Candida condition, in particular Candida
hyphal growth. Dosages and administration of an antagonist of the
invention in a pharmaceutical composition may be determined by one of
ordinary skill in the art of clinical pharmacology or pharmacokinetics.
See, for example, Mordenti and Rescigno, (1992) Pharmaceutical Research,
9:17-25; Morenti et al., (1991) Pharmaceutical Research, 8:1351-1359; and
Mordenti and Chappell, "The use of interspecies scaling in
toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al.
(eds) (Pergamon Press: NY, 1989), pp. 42-96. An effective amount of the
antagonist to be employed therapeutically will depend, for example, upon
the therapeutic objectives, the route of administration, and the
condition of the mammal. Accordingly, it will be necessary for the
therapist to titer the dosage and modify the route of administration as
required to obtain the optimal therapeutic effect. A typical daily dosage
might range from about 10 ng/kg to up to 100 mg/kg of the mammal's body
weight or more per day, preferably about 1 μg/kg/day to 10 mg/kg/day.

[0035]Preferably, the antagonist interferes with Candida hyphal growth by
means that affect the concentration or presence of or binding activity of
muramyl-dipeptide-related compounds in body fluids (such as without
limitation, blood, plasma, bodily fluids in esophagus, throat,
interstitial fluid, lymph, mucus, etc) to the LRR-domain in CaCdc35p.
More preferably, the antagonist is effective against
muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanine,
N-acetylmuramyl-L-alanyl-D-isoglutamine or
muramyl-L-alanyl-L-isoglutamine. In a highly preferred form, the
antagonist is specifically effective against
muramyl-L-alanyl-D-isoglutamine.

[0036]The term "antagonist" is used in the broadest sense, and includes
any molecule that partially or fully blocks, inhibits, or neutralizes a
biological activity of muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure. Such antagonists may work by
engaging either muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure or they may engage the LRR
domain of CaCdc35p preventing muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in their core structure from binding to the LRR
domain.

[0037]Suitable antagonist molecules specifically include antagonist
antibodies or antibody fragments, muramyl-dipeptide analogues of
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure, and small organic molecules, etc.

[0038]Methods for identifying antagonists of a muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure may
comprise contacting a muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure with the LRR domain of CaCdc35p
in the presence of a candidate agonist or antagonist molecule and
measuring hyphal growth.

[0039]Consistent with the invention there are provided (a) antibodies to
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure and (b) antibodies that engage the LRR domain of
CaCdc35p preventing muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure from binding to the LRR domain.
Exemplary antibodies include polyclonal, monoclonal, humanized,
bispecific, and heteroconjugate antibodies.

[0040]A. Polyclonal Antibodies

[0041]The antibodies of the invention may comprise polyclonal antibodies.
Methods of preparing polyclonal antibodies are known to the skilled
artisan. Polyclonal antibodies can be raised in a mammal, for example, by
one or more injections of an immunizing agent and, if desired, an
adjuvant.

[0042]Typically, the immunizing agent and/or adjuvant will be injected in
the mammal by multiple subcutaneous or intraperitoneal injections. The
intensity of the response is determined by several factors including the
size of the immunogen molecule, its chemical characteristics, and how
different it is from the animal's own proteins. Most natural immunogens
are proteins with a molecular weight above 5 kDa that come from sources
phylogenically far removed from the host animal (i.e., human proteins
injected into rabbits or goats). It is desirable to use highly purified
proteins as immunogens, since the animal will produce antibodies to even
small amounts of impurities present as well as to the major component.
The antibody response increases with repeated exposure to the immunogen,
so a series of injections at regular intervals is needed to achieve both
high levels of antibody production and antibodies of high affinity.

[0043]To the extent that the antagonist is an antibody that engage the LRR
domain of CaCdc35p preventing muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in their core structure from binding to the LRR
domain the immunogen will be an selected from amino acids comprising the
LRR domain from CaCdc35p. Preferably, the amino acid sequence will be
selected from the region of about 363 to 927 in the CaCdc35p protein.
Sequences of at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 amino acids from
this region will generally be used to generate those antibodies.
Desirably, the sequence selected will generate an antibody that
specifically interferes with binding of muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure to the
LRR domain of only CaCdc35p.

[0044]Not all immunogenic molecules will however generate the level of
antibody desired. To increase the intensity of the immune response
immunogens are combined with complex mixtures called adjuvants. Adjuvants
are a mixture of natural or synthetic compounds that, when administered
with antigens, enhance the immune response. Adjuvants are used to (1)
stimulate an immune response to an antigen that is not inherently
immunogenic, (2) increase the intensity of the immune response, (3)
preferentially stimulate either a cellular or a humoral response (i.e.,
protection from disease versus antibody production). Examples of
adjuvants which may be employed include Freund's complete adjuvant and
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate). A more extensive discussion of adjuvants and their use
in immunization protocols is given in Immunology Methods Manual, vol. 2,
I. Lefkovits, ed., Academic Press, San Diego, Calif., 1997, ch. 13.
Immunology Methods Manual is available as a four volume set, (Product
Code Z37, 435-0); on CD-ROM, (Product Code Z37, 436-9); or both, (Product
Code Z37, 437-7)

[0045]If the immunogen is still unable to generate an acceptable response,
it may be conjugated to a carrier protein that is more immunogenic. Small
molecules such as drugs, organic compounds, and peptides and
oligosaccharides with a molecular weight of less than 2-5 kDa like, for
example, muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure, are not usually immunogenic,
even when administered in the presence of adjuvant. In order to generate
an immune response to these compounds, it is necessary to attach them to
a protein or other compound, termed a carrier that is immunogenic. When
attached to a carrier protein the small molecule immunogen is called a
hapten. Haptens are also conjugated to carrier proteins for use
immunoassays. The carrier protein provides a means of attaching the
hapten to a solid support such as a microtiter plate or nitrocellulose
membrane. When attached to agarose they may be used for purification of
the anti-hapten antibodies. They may also be used to create a multivalent
antigen that will be able to form large antigen-antibody complexes. When
choosing carrier proteins, remember that the animal will form antibodies
to the carrier protein as well as to the attached hapten. It is therefore
relevant to select a carrier protein for immunization that is unrelated
to proteins that may be found in the assay sample. If haptens are being
conjugated for both immunization and assay, the two carrier proteins
should be as different as possible. This allows the antiserum to be used
without having to isolate the anti-hapten antibodies from the
anti-carrier antibodies.

[0046]Where the immunizing agent is muramyl-L-alanine and/or compounds
that include muramyl-L-alanine in their core structure preferably the
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure are conjugated to a protein known to be immunogenic
in the mammal being immunized.

[0047]Examples of such immunogenic proteins include but are not limited to
keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin,
soybean trypsin inhibitor, and a toxoid, for example tetanus toxoid.

[0048]KLH is a respiratory protein found in mollusks. Its large size makes
it very immunogenic, and the large number of lysine residues available
for conjugation make it very useful as a carrier for haptens. The
phylogenic separation between mammals and mollusks increases the
immunogenicity and reduces the risk of cross-reactivity between
antibodies against the KLH carrier and naturally occurring proteins in
mammalian samples.

[0049]KLH is offered both in its native form, for conjugation via amines,
and succinylated, for conjugation via carboxyl groups. Succinylated KLH
may be conjugated to a hapten containing amine groups (such as a peptide)
via cross-linking with carbodiimide between the newly introduced carboxyl
groups of KLH and the amine groups of the hapten.

[0051]The immunization protocol may be selected by one skilled in the art
without undue experimentation. Protocols for preparing immunogens,
immunization of animals, and collection of antiserum may be found in
Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring
Harbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp. 55-120 (Product
Code A 2926).

[0052]B. Monoclonal Antibodies

[0053]The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such as
those described by Kohler and Milstein (1975), Nature, 256:495. In a
hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized with an immunizing agent as described above to elicit
lymphocytes that produce or are capable of producing antibodies that will
specifically bind to the immunizing agent. Alternatively, the lymphocytes
may be immunized in vitro.

[0054]Generally, either peripheral blood lymphocytes ("PBLs") are used if
cells of human origin are desired, or spleen cells or lymph node cells
are used if non-human mammalian sources are desired. The lymphocytes are
then fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell. Immortalized cell
lines are usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine and human origin. Usually, rat or mouse myeloma cell
lines are employed. The hybridoma cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will include hypoxanthine, aminopterin, and
thymidine ("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.

[0055]Preferred immortalized cell lines are those that fuse efficiently,
support stable high level expression of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. More preferred immortalized cell lines are murine myeloma lines,
which can be obtained, for instance, from the Salk Institute Cell
Distribution Center, San Diego, Calif. and the American Type Culture
Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma
cell lines also have been described for the production of human
monoclonal antibodies (Kozbor, J. (1984) Immunol., 133:3001).

[0056]The culture medium in which the hybridoma cells are cultured can
then be assayed for the presence of monoclonal antibodies directed
against muramyl-L-alanine and/or compounds that include muramyl-L-alanine
in their core structure.

[0057]After the desired hybridoma cells are identified, the clones may be
subcloned by limiting dilution procedures and grown by standard methods.
Suitable culture media for this purpose include, for example, Dulbecco's
Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the
hybridoma cells may be grown in vivo as ascites in a mammal.

[0058]The monoclonal antibodies secreted by the subclones may be isolated
or purified from the culture medium or ascites fluid by conventional
immunoglobulin purification procedures such as, for example, protein
A-Sepharose, hydroxylapatite chromatography, gel electrophoresis,
dialysis, or affinity chromatography.

[0059]The monoclonal antibodies may also be made by recombinant DNA
methods, such as those described in U.S. Pat. No. 4,816,567.

[0060]The antibodies may be monovalent antibodies. Methods for preparing
monovalent antibodies are well known in the art. For example, one method
involves recombinant expression of immunoglobulin light chain and
modified heavy chain. The heavy chain is truncated generally at any point
in the Fc region so as to prevent heavy chain cross-linking.
Alternatively, the relevant cysteine residues are substituted with
another amino acid residue or are deleted so as to prevent cross-linking.

[0061]In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine techniques
known in the art.

[0062]C. Human and Humanized Antibodies

[0063]The antibodies of the invention may further comprise humanized
antibodies or human antibodies. Humanized forms of non-human (e.g.,
murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other
antigen-binding sub-sequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized antibodies
include human immunoglobulins (recipient antibody) in which residues from
a complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as
mouse, rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework sequences.
In general, the humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are those
of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin.

[0064]Methods for humanizing non-human antibodies are well known in the
art. Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These non-human
amino acid residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization can be
essentially performed following the method of Winter and co-workers
[Jones et al., (1986) Nature, 321:522-525; Riechmann et al., (1988)
Nature, 332:323-327; Verhoeyen et al., (1988) Science 239:1534-1536], by
substituting rodent CDRs or CDR sequences for the corresponding sequences
of a human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR residues and
possibly some FR residues are substituted by residues from analogous
sites in rodent antibodies.

[0067]Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding specificities for at least two different
antigens. In the present case, one of the binding specificities is for
muramyl-L-alanine and/or a compound that includes muramyl-L-alanine in
its core structure, the other one is for another compound having
muramyl-L-alanine in its core structure.

[0068]Methods for making bispecific antibodies are known in the art.
Traditionally, the recombinant production of bispecific antibodies is
based on the co-expression of two immunoglobulin heavy-chain/light-chain
pairs, where the two heavy chains have different specificities [Milstein
and Cuello, (1983) Nature, 305:537-539].

[0069]E. Heteroconjugate Antibodies

[0070]Heteroconjugate antibodies are also within the scope of the present
invention. Heteroconjugate antibodies are composed of two covalently
joined antibodies. Such antibodies have, for example, been proposed to
target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],
and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089].
It is contemplated that the antibodies may be prepared in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond. Examples of
suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Pat. No. 4,676,980.

[0071]F. Immunoconjugates

[0072]The invention also pertains to immunoconjugates comprising an
antibody conjugated to a cytotoxic agent such as a chemotherapeutic
agent, toxin (e.g., an enzymatically active toxin against Candida), or a
radioactive isotope (i.e., a radioconjugate).

[0074]Antibodies produced according to the invention, as well as other
molecules identified by the screening assays disclosed herein, can be
administered for the treatment of Candida infection in the form of
pharmaceutical compositions.

[0075]Thus, the present invention also relates to compositions including
pharmaceutical compositions comprising a therapeutically effective amount
of an antagonist to muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure. As used herein a compound will
be therapeutically effective if it is able to affect Candida hyphal
growth.

[0076]Pharmaceutical forms of the invention suitable for injectable use
include sterile aqueous solutions (where water soluble) or dispersions
and sterile powders for the extemporaneous preparation of sterile
injectable solutions and or one or more carrier. Alternatively,
injectable solutions may be delivered encapsulated in liposomes to assist
their transport across cell membrane. Alternatively or in addition such
preparations may contain constituents of self-assembling pore structures
to facilitate transport across the cellular membrane. It must be stable
under the conditions of manufacture and storage and must be preserved
against the contaminating/destructive action of microorganisms such as,
for example, bacteria and fungi.

[0077]The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol
and liquid polyethylene glycol, and the like), suitable mixtures thereof,
and vegetable oils. The proper fluidity can be maintained, for example,
by the use of a coating such as, for example, lecithin, by the
maintenance of the required particle size in the case of dispersion and
by the use of surfactants. Preventing the action of microorganisms in the
compositions of the invention is achieved by adding antibacterial and/or
antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic
acid, thimerosal and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about
by the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.

[0078]Sterile injectable solutions are prepared by incorporating the
active compounds in the required amount in the appropriate solvent with
several of the other ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredient into a sterile
vehicle which contains the basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum drying and freeze-drying, to yield a
powder of the active ingredient plus any additional desired ingredient
from previously sterile-filtered solution thereof.

[0079]When the active ingredients, in particular small molecules
contemplated within the scope of the invention, are suitably protected
they may be orally administered, for example, with an inert diluent or
with an edible carrier, or it may be enclosed in hard or soft shell
gelatin capsule, or it may be compressed into tablets, or it may be
incorporated directly with the food of the diet. For oral therapeutic
administration, the active compound may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% by weight of
active compound. The percentage of the compositions and preparations may,
of course, be varied and may conveniently be between about 5 to about 80%
of the weight of the unit. The amount of active compound in such
therapeutically useful compositions in such that a suitable dosage will
be obtained. Preferred compositions or preparations according to the
present invention are prepared so that a dosage unit form contains
between about 0.1 μg and 20 g of active compound.

[0080]The tablets, troches, pills, capsules and the like may also contain
binding agents, such as, for example, gum, acacia, corn starch or
gelatin. They may also contain an excipient, such as, for example,
dicalcium phosphate. They may also contain a disintegrating agent such
as, for example, corn starch, potato starch, alginic acid and the like.
They may also contain a lubricant such as, for example, magnesium
stearate. They may also contain a sweetening agent such a sucrose,
lactose or saccharin. They may also contain a flavouring agent such as,
for example, peppermint, oil of wintergreen, or cherry flavouring.

[0081]When the dosage unit form is a capsule, it may contain, in addition
to materials of the above type, a liquid carrier.

[0082]Various other materials may be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar or both. A syrup or
elixir may contain the active compound, sucrose as a sweetening agent,
methyl and propylparaben as preservatives, a dye and flavouring such as,
for example, cherry or orange flavour. Of course, any material used in
preparing any dosage unit form should be pharmaceutically pure and
substantially non-toxic in the amounts employed. In addition, the active
compound(s) may be incorporated into sustained-release preparations and
formulations.

[0083]The present invention also extends to forms suitable for topical
application such as, for example, creams, lotions and gels. Such a
formulation comprises a gelling agent in a concentration effective to
promote gelling upon contact with the eye or with lacrimal fluid in the
exterior of the eye. Suitable gelling agents include, but are not limited
to, thermosetting polymers such as tetra-substituted ethylene diamine
block copolymers of ethylene oxide and propylene oxide (e.g.,
poloxamine); polycarbophil; and polysaccharides such as gellan,
carrageenan (e.g., kappa-carrageenan and iota-carrageenan), chitosan and
alginate gums.

[0084]To this extent the active ingredient may be held within a matrix
which controls the release of the active agent. Preferably, the matrix
comprises a substance selected from the group consisting of lipid,
polyvinyl alcohol, polyvinyl acetate, polycaprolactone,
poly(glycolic)acid, poly(lactic)acid, polycaprolactone, polylactic acid,
polyanhydrides, polylactide-co-glycolides, polyamino acids, polyethylene
oxide, acrylic terminated polyethylene oxide, polyamides, polyethylenes,
polyacrylonitriles, polyphosphazenes, poly(ortho esters), sucrose acetate
isobutyrate (SAIB), and combinations thereof and other polymers such as
those disclosed in U.S. Pat. Nos. 6,667,371; 6,613,355; 6,596,296;
6,413,536; 5,968,543; 4,079,038; 4,093,709; 4,131,648; 4,138,344;
4,180,646; 4,304,767; 4,946,931, each of which is expressly incorporated
by reference herein in its entirety. Preferably, the matrix sustainedly
releases the drug.

[0085]Pharmaceutically acceptable carriers and/or diluents may also
include any and all solvents, dispersion media, coatings, antibacterials
and/or antifungals, isotonic and absorption delaying agents and the like.
The use of such media and agents for pharmaceutical active substances is
well known in the art. Except insofar as any conventional media or agent
is incompatible with the active ingredient, use thereof in the
therapeutic compositions is contemplated.

[0087]It is especially advantageous to formulate parenteral compositions
in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units
suited as unitary dosages for the mammalian subjects to be treated, each
unit containing a predetermined quantity of active material calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier. The dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique characteristics of
the active material and the particular therapeutic effect to be achieved,
and (b) the limitations inherent in the art of compounding such an active
material for the treatment of disease in living subjects having a
diseased condition in which bodily health is impaired as herein disclosed
in detail.

[0088]The principal active ingredient is compounded for convenient and
effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form. A unit dosage
form can, for example, contain the principal active compound in amounts
ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the
active compound is generally present in from about 0.5 pg to about 2000
mg/ml of carrier. In the case of compositions containing supplementary
active ingredients, the dosages are determined by reference to the usual
dose and manner of administration of the said ingredients.

Prognosing or Diagnosing the Course of a Candida Infection

[0089]The invention also provides a means for prognosing or diagnosing the
course of a Candida infection, comprising the steps of: measuring the
amount of muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure in body fluids sampled from a
Candida infection.

[0090]Further the invention provides a means for determining the most
effective treatment for a Candida infection, comprising the steps of:
diagnosing the state of hyphal growth according to the above method and
then determining the patient's treatment according to whether hyphal
growth is expected or not.

[0091]Diagnostic and prognostic methods will generally be conducted using
a biological sample obtained from a patient. A "sample" refers to a
sample of tissue or fluid suspected of containing an muramyl-L-alanine
and/or compounds that include muramyl-L-alanine in their core structure
from an individual including, but not limited to, e.g., plasma, serum,
spinal fluid, lymph fluid, the external sections of the skin,
respiratory, intestinal, and genitourinary tracts, tears, saliva, blood
cells, tumours, organs, tissue and samples of in vitro cell culture
constituents.

[0092]According to the diagnostic and prognostic methods of the present
invention, alteration of levels of muramyl-L-alanine and/or compounds
that include muramyl-L-alanine in their core structure in body fluids may
be detected using anyone of the methods described herein.

[0093]Alteration of levels of muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in their core structure in body fluids can be
detected by screening for such compounds. Such alterations can be
determined by any assay that detects changes in the level of
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure such as biochemical assays like HLPC assays and the
like or immunological assays in accordance with conventional techniques.
Antibodies (polyclonal or monoclonal) as described herein may be used to
detect muramyl-L-alanine and/or compounds that include muramyl-L-alanine
in their core structure in body fluids. The antibodies may be prepared as
discussed above. Immunological assays can be done in any convenient
format known in the art. These include Western blots, immunohistochemical
assays and ELISA assays. Any means for detecting a binding pair can be
used. Functional assays, such as protein binding determinations, can be
used.

Screening for Agonists and Antagonists of Candida Hyphal Growth

[0094]Consistent with the invention there is provided a means for
screening for agonists and antagonists of Candida hyphal growth
comprising the steps of: (a) contacting (i) muramyl-L-alanine and/or
compounds that include muramyl-L-alanine in their core structure and (ii)
the LRR domain of CaCdc35p from a Candida species with a sample compound,
and (b) detecting whether the sample compound exhibits agonistic or
antagonistic activity towards the interaction. Preferably, the method is
used to screen for antagonistic drugs that are capable of interfering
either in a direct or indirect manner with the interaction between C.
albicans and muramyl-L-alanine, muramyl-L-alanyl-D-isoglutamine,
N-acetyl-muramyl-L-alanine, N-acetyl-muramyl-L-alanyl-D-isoglutamine, or
muramyl-L-alanyl-L-isoglutamine.

[0095]Screening assays for antagonist drug candidates are designed to
identify compounds that bind the LRR domain of CaCdc35p to inhibit the
binding of muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure with the protein or interfere
with the interaction between muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in their core structure and the LRR domain of
CaCdc35p. Such screening assays will include assays amenable to
high-throughput screening of chemical libraries, making them particularly
suitable for identifying small molecule drug candidates.

[0096]The assays can be performed in a variety of formats, including
protein binding assays, biochemical screening assays, immunoassays, and
cell-based assays, which are well characterized in the art. Such assays
for antagonists are common in that they call for contacting
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure and the LRR domain of CaCdc35p in the presence of
the drug candidate for a time sufficient to allow these components to
interact.

[0097]Compounds that interfere with the interaction can be tested as
follows: usually a reaction mixture is prepared containing
muramyl-L-alanine and/or compounds that include muramyl-L-alanine in
their core structure and at least the LRR domain of CaCdc35p for a time
allowing for the interaction and binding of the products. To test the
ability of a candidate compound to inhibit binding, the reaction is run
in the absence and in the presence of the test compound. In addition, a
placebo may be added to a third reaction mixture, to serve as positive
control. The binding (complex formation) between the test compound and
the intra- or extracellular components present in the mixture is
monitored as described hereinabove. The formation of a complex in the
control reaction(s) but not in the reaction mixture containing the test
compound indicates that the test compound interferes with the interaction
of the test compound and its reaction partner.

[0098]Potential antagonists include small molecules that bind to the site
in the LRR domains where muramyl-L-alanine and/or compounds that include
muramyl-L-alanine in their core structure bind, thereby blocking the
normal biological activity of muramyl-L-alanine and/or compounds that
include muramyl-L-alanine in their core structure. Examples of small
molecules include, but are not limited to, small peptides or peptide-like
molecules, preferably soluble peptides, and synthetic non-peptidyl
organic or inorganic compounds.

[0099]These small molecules can be identified by any one or more of the
screening assays discussed hereinabove and/or by any other screening
techniques well known for those skilled in the art.

[0100]The present invention also relates to compounds identified by the
above method and their use in treating Candida hyphal growth in a
patient.

Non-Limiting Illustration of the Invention

[0101]Further features of the present invention are more fully described
in the following description. This description is included solely for the
purposes of exemplifying the present invention. It should not be
understood as a restriction on the broad description of the invention as
set out above.

[0102]The following discussion describes the identification and functional
characterization of the hyphal inducer(s) in human and bovine sera and
their sensor in C. albicans. It shows that peptidoglycan-like molecules
are significantly enriched in the chromatographic fractions of serum with
strong hypha-inducing activity. Through chemical synthesis, the inventor
has found that compounds with a core structure of muramyl-L-alanine
(e.g.: FIG. 1) are potent hyphal inducers with
muramyl-L-alanine-D-isoglutamine being the most active. They also show
that the inducers may directly bind the LRR domain of the adenylate
cyclase Cdc35, stimulate cAMP production and promote hyphal growth.

Co-Precipitation of Hypha-Inducing Activity from Serum with Serum Proteins

[0103]Precipitation of serum proteins with acetonitrile trapped about
-60-70% of the hypha-inducing activity in the protein pellet and a brief
treatment of serum at room temperature with weak acid, such as 1%
trifluoroacetic acid (TFA), was sufficient to release a majority of the
activity into supernatant (FIG. 2). The results indicate that a majority
of the hypha-inducing agents, in serum is present in protein-bound form.
Hence, in the fractionation procedure described below the bovine and
human sera were first treated with 1% TFA before filtration through a
membrane with a molecular weight cut off of 3 kDa to remove serum
proteins. Fast performance liquid chromatography (FPLC) fractionation of
the serum filtrate detected significant hypha-inducing activity in a
single peak centered about fraction 30 (Ff) (FIG. 3, left). Ff30 was
highly active in hyphal induction, inducing 50% germ tube formation
(I50) at -0.2 mg (dry weight)/ml. In comparison, fresh serum and the
<3 kDa filtrate had I50 values of -5.5 and 1.3 mg/ml
respectively. Further separation of Ff30 by using conventional
reversed-phase high performance liquid chromatography (HPLC) found the
hypha-inducing activity to be relatively evenly present in fractions 5 to
10. To achieve better separation, the active fractions 5 to 10 were
pooled, freeze-dried and subjected to a. second round of reversed-phase
HPLC using a Waters Atlantis dC18, 5 μM column. This column allows the
use of aqueous mobile phase and provides much improved retention of polar
compounds. The hyphal induction assay located high hypha-inducing
activity in fractions with retention times from 10 and 14 min
corresponding to a group of peaks with low UV absorption (FIG. 3). The
active fractions (Hf 10/14), when pooled, exhibited an I50 of
approximately 0.12 mg/ml. The hypha-inducing activity of both human and
bovine sera exhibited nearly identical chromatographic profiles and
retention times, indicating that the inducers are similar in nature.
Attempts to further resolve the active fractions by using a range of
sizing, ion exchange and affinity chromatography were not successful,
because the activity was always distributed rather broadly. Inventor
obtained a total of approximately 7 mg of dry material of Hf10/14 from
500 ml serum and subjected it for NMR analysis. The NMR spectra cleared
showed signals for glucose, fructose, glycerol and lactic acid. However,
none of these compounds was found to have appreciable hypha-inducing
activity individually or in combination in PBS or Hank's solution.
However, further analysis of the low intensity NMR signals suggested the
presence of muramic acid, alanine and isoglutamine. However, owing to the
weak signals and impurity of the sample, there was uncertainty whether
the three moieties belong to the same molecule. Intriguingly, muramic
acid (Mur) is thought to be only present in bacterial PGs in nature; and
alanine and isoglutamine are very common amino acids, of the short
peptides cross-linking the N-acetylglucosamine-N-acetylmutamic acid
chains in PGs (FIG. 1). Thus, two chemical structures have been
identified: muramyl-L-alanine (FIG. 1 top) and
muramyl-L-alanyl-D-isoglutamine (FIG. 1 bottom and FIG. 4). Inventor also
noted that in nature muramic acid is almost universally found in
2-N-acetyl form, but their NMR data did not detect this group in the
proposed muramic acid.

The Presences of Mur-Containing Compounds in the Active Serum Fractions

[0104]Although Mur-containing compounds have been detected in a range of
normal and inflammatory tissues of mammals (including brain, kidney,
liver and peripheral leukocytes and in urine), its presence in serum
remains controversial.

[0105]Established protocols were used to confirm the presence of
Mur-containing molecules in serum and their enrichment in the
chromatographic fractions active for hyphal induction. To release free
Mur, samples were first hydrolyzed with 4N HCl and then reduced by sodium
borohydride to remove the anomeric center of the ring that is well known
to cause the splitting of chromatographic peaks. To enhance detection
sensitivity, the reduced samples were derivatized by dansylation, which
adds a fluorescent dansyl group to each free NH2 group. The
dansylated samples were separated by reversed-phase HPLC using a Waters
Sunfire C18 column. The UV spectra of Hf10/14 processed by following the
above protocol showed a well isolated peak with a retention time of 22.07
min that matches that of the authentic Mur processed in an identical
fashion (FIG. 3). Mass spectrometry (MS) analysis of this peak of the
derivatized authentic Mur revealed a clean spectrum with two prominent
ions of m/z 487.1 (M+H).sup.+ and 469.1 that are consistent with dansyl
Mur and dansyl Mur minus one H2O respectively. MS analysis of the
peak from Hf10/14 produced both 487.1 and 469.1 ions. To gain more robust
results borohydride was replaced with sodium borodeuteride in the
reduction step, which is expected to increase the mass of the dansylated
Mur to 488.1. Under this condition, HPLC detected the same peak at 22.07
min and MS analysis of the peak revealed strong ions of m/z 488.1 and
470.1. By contrast, the Mur signals were not detected if the HCl
hydrolysis step was omitted; indicating that serum Mur is predominantly
present as a moiety of larger PG fragments. Furthermore, significant Mur
signals were not detected from any of the serum fractions without
hypha-inducing activity, indicating that the majority of serum
Mur-containing compounds are concentrated in the chromatographic
fractions with strong hypha-inducing activity. Using this protocol Mur
was invariably detected from several different batches of bovine sera, 2
human plasma samples and 10 human sera. Mur was not detected from mock
samples (water or PBS) processed by following the same procedure,
excluding the possibility of bacterial contamination. When the <3 kDa
serum filtrate was directly processed by this protocol, the HPLC peak for
dansyl Mur overlapped with high chemical complexity of the filtrate, made
the detection difficult. This observation might explain, at least in
part, why some earlier efforts failed to detect Mur in serum. In summary,
the NMR data suggested the presence of muramyl-L-alanyl-D-isoglutamine in
the serum fractions enriched for hypha-inducing activity; and
subsequently MS analysis confirmed a significant enrichment of
Mur-containing molecules and detected a MS ion corresponding to the mass
of muramyl-alanyl-isoglutamine in the same fractions. This is believed to
be the first unequivocal detection of Mur-containing molecules in sera
from healthy people and animals. Using authentic muramic acid as a
standard in HPLC, the total amount of muramic acid detected in the FPLC
fractions Hf31 to 33 was quantified. These data established that the
amount of Mur in both human and bovine serum is at least 0.5-1 mM.

Synthetic PG Components Exhibited Potent Hypha-Inducing Activity

[0106]To determine whether the NMR-elucidated compound is active for
hyphal induction, solid-phase chemical synthesis was undertaken of
muramyl-L-alanyl-D-isoglutamine (MLADiQ) and muramyl-L-alanine (MLA).
N-acetylmuramyl-L-alanyl-D isoglutamine (NMLADiQ) and
N-acetylmurmyl-L-alanine (NMLA) was also synthesized together with
compounds containing D-alanine (MDA and MDADiQ) and L-isoglutamine
(MLALiQ) to evaluate the, importance of the N-acetyl group and the
stereo-chemical configuration of the amino acids for hyphal induction.
The schemes for chemical synthesis of the compounds followed standard
procedures. All the synthesized compounds were purified by HPLC and their
identities confirmed by MS. The I50 of each compound was then
determined. These data revealed that MLADiQ was a highly potent hyphal
inducer with an I50 of approximately 10 μM. Strikingly, the
N-acetylated compound NMLADiQ had an I50 of approximately 6 mM, 600
times less active than MLADiQ. Also, NMLADiQ at increased concentrations
was never able to induce higher than 60% germ tube formation and the
activity started to drop significantly when the concentration was raised
above 20 mM. MLA was also active with an I50 of approximately 200
μM, while NMLA had an I50 of approximately 8 mM and exhibited
similar diminishing hypha-inducing activity at high concentrations as
NMLADiQ. The compounds with the L-alanine substituted by D-alanine (MDA
and MDADiQ) were inactive for hyphal induction, whereas the compound with
the D-isoglutamine replaced by L-isoglutamine (MLALiQ) exhibited reduced
but still substantial activity with an I50 of approximately 180
μM. Neither muramic nor N-acetylmuramic acid was active for hyphal
induction. N-Acetylglucosamine (NAG), the strongest single-compound
hyphal inducer previously known had an I50 of approximately 3 mM.
Glucose had no activity in the assay conditions. Taken together, the
following conclusions can be drawn from these data. First, MLADiQ is a
highly potent hyphal inducer, consistent with the NMR-- elucidated
chemical structure. Second, the absence of the, N-acetyl group in the
muramyl moiety is crucial for high hypha-inducing activity, which
provides an excellent explanation about why our NMR' analysis did not
detect signals for the N-acetyl group. Hyphin of FIG. 1 does not have the
N-acetyl group commonly found in bacterial PGNs. Third, muramyl-L-alanine
appears to be the minimal structure required and its L configuration is
essential for the hypha-inducing activity. Fourth, the results suggest
that other PG components structurally related to MLADiQ may also be
active for hyphal induction.

The Role of the LRR Domain of C. Albicans Adenylate Cyclase CaCdc35p

[0107]Pathogen-associated molecular patterns, including PG motifs, are
known to be recognized by LRR domain-containing proteins in mammals,
plants and Drosophila to initiate host innate immune response. In view of
these data studies were carried out to determine if C. albicans might use
a similar mechanism for MLADiQ sensing.

[0108]BLAST-searches were conducted to on the C. albicans genome database
to identify proteins that may contain LRR domain-containing proteins.
Data from these searches lead to the identification of a single
significant match with CaCdc35p. CaCdc35p is a large protein of 1690
amino acids (aa) with multiple functional domains: a Ras association (RA)
domain (aa 304-393), 15 LRRs (aa 490-927) organized in three clusters,
and an adenylyl cyclase (CYCc) domain (aa 12471500). CaCdc35p has been
positioned near the top of the cAMPIPKA signal transduction pathways for
hyphal growth (Leberer, E., et al. (2001) Mol. Microbiol. 42, 673-687;
Roche, C. R., et al (2001) Mol. Biol. Cell 12, 3631-3643). CaCDC35
deletion mutant is completely blocked for hyphal development and exhibits
severely retarded yeast growth as well (Leberer, E., et al. (2001) Mol.
Microbiol. 42, 673-687). To assess whether CaCdc35p LRR domain is
required for sensing MLADiQ, a series of cacdc35 mutants deleted of
either the entire LRR domain (IrrΔ) or each of the three LRR
clusters (Irr1Δ, Irr2Δ and Irr3Δ) were created. Point
mutations were also introduced in some of the highly conserved residues
within a repeat. For example, mutants Irr5mu and Irr9mu carrying
Leu→Ala and Asn→Ala mutations in repeats 5, and 9
respectively. The genomic DNA fragment encoding CaCdc35p and
approximately 500 bp of both 5' and 3' flanking sequences was cloned in
plasmid Clp10 as the template for mutation. Each of the constructs was
integrated at a specific site in the promoter region of the CaCDC35locus
in a cacdc35A mutant and the expression was verified by Western blot
analysis. The wild-type CaCDC35 fully rescued the hyphal development
defect of cacdc35Δ in response to MLADiQ as well as serum and
reduced the doubling time of yeast growth from 4.46 h to 1.67 h in GMM at
30° C. Strikingly, although all LRR domain mutants largely rescued
the retarded yeast growth of cacdc35Δ, none restored to any extent
the hyphal development in response to MLADiQ and serum. The results
suggest that the LRR domain may have a specific role in mediating the
hypha-inducing signals and this activity is largely separable from the
general growth function of the protein.

[0109]One crucial early event in C. albicans hyphal growth is the
occurrence of a spike of intracellular cAMP. The observation that the LRR
domain mutants rescued the yeast growth defect of cacdc35A but not the
hyphal growth defect suggests that the mutated CaCdc35p may be able to
provide a basal level of cellular cAMP which is important for general
growth functions but unable to increase cAMP production which is required
for the activation of the cAMP/PKA pathway. To test this hypothesis, cAMP
levels were examined in wild-type and several LRR domain mutants in
response to MLADiQ treatment. The yeast cells were treated with 50 μM
MLADiQ, a concentration that consistently induced near 100% yeast-hypha
switch in wild-type strains, and aliquots were harvested every 20 min for
cAMP assay. In the wild-type yeast cells the intracellular cAMP level was
found to be approximately 1.7 pmol/mg dry cells. Upon hyphal induction by
MLADiQ at 37° C. the cAMP level rapidly rose to 3.8 by 30 to 40
min, gradually declined in the next 30 min to approximately 2.8 pmol and
remained at this level during hyphal growth. By contrast, cAMP was
undetectable in cacdc35Δ cells. Re-introducing in the mutant a copy
of CaCDC35, IrrΔ or Irr9mu restored the CAMP level to 1.2-1.7
pmol/mg dry cells during yeast growth. However, the MLADiQ-induced cAMP
spike was only detected in the cacdc35Δ cells transformed with
CaCDC35. The results demonstrate that the LRR domain mutants can provide
a basal level of cellular cAMP but unable to increase it in response to
MLADiQ treatment.

[0110]Next, assays were performed for the conversion of
α-32P-ATP to α-32P-cAMP in cell lysate after the
addition of MLADiQ, which is a direct measure of the adenylate cyclase
activity. Cell lysate was prepared by glass bead-beating of cells under
nondenaturing conditions. Adding MLADiQ to the wild-type lysate activated
a32P-cAMP production and as low as approximately 6 μM of MLADiQ was
sufficient to induce the maximum level of activation of the catalytic
activity. By contrast, no increase of the cyclase activity was observed
in the lysates of IrrΔ and Irr9mu cells. The data indicate that
MLADiQ directly enhances the adenylate cyclase activity of CaCdc35p in an
LRR domain-dependent manner.

Direct Interaction Between Recombinant CaCdc35p LRR Domain and MLADiQ

[0111]Next, the possibility that MLA 7IQ may directly bind to the LRR
domain of CaCdc35p was explored. The wild-type LRR domain and that of
Irr9mu were expressed in E. coli as glutathione-S-transferase (GST)
fusions and purified to high homogeneity. Then circular dichroism (CD)
spectroscopy was used to detect possible direct interaction between the
LRR domain and MLADIQ. CD is a reliable tool in detecting conformational
changes in a protein as a result of ligand binding (Woody, R. W. (1995).
Methods Enzymol. 246, 34-71). Adding MLADiQ to the GST-LRR fusion protein
resulted in a significant concentration-dependent shift of the
ellipticity values of the CD spectra, whereas this effect was not
observed on the spectra of GST-Irr9mu and GST. None of the compounds
lacking the hypha-inducing activity exhibited significant effect on the
CD spectra of LRR, whereas the less active hyphal inducers MLA, NMLADiQ
and NAG caused a weaker but consistent spectrum shift. Together, the
results of both the fluorescence and CD spectroscopy demonstrate a direct
interaction between MLADiQ and the LRR domain of CaCdc35p as well as an
excellent correlation between the hypha-inducing activity of the
components and their affinity for LRR binding.

[0112]Since specific binding of a molecule to a tryptophan-containing
protein may cause concentration-dependent quenching; of tryptophan
fluorescence, fluorescence spectroscopy was used to measure the intensity
of tryptophan fluorescence of the recombinant LRR domains in the presence
of different concentrations of MLADiQ. The results showed that MLADiQ
caused significant fluorescence quenching of the GST-LRR fusion in a
concentration dependent fashion. In comparison, MDADiQ did not cause any
considerable quenching, whereas the less potent inducers MLA, NMLADiQ and
NAG induced lower levels of fluorescence quenching. The fluorescence
spectrum of GST-Irr9mu appeared similar to that of GST-LRR in the absence
of ligand, suggesting that the point mutations had little effect on the
overall conformation of the domain. However, the mutated domain did not
exhibit detectable fluorescence quenching when mixed with MLADIQ. The
fluorescence spectrum of GST was not affected by any of the compounds
used. Nonlinear regression analysis of the fluorescence quenching as a
function of MLADiQ concentration suggested unimodal binding with an
equilibrium dissociation constant (Kd) of 1.44±0.14 μM and a
1:1 ligand/receptor interaction. MLA, NMLADiQ and NAG exhibited Kd
values of 1.67±0.13, 5.26±0.47 and 6.80±0.32 μM respectively.

[0113]Modifications of the above-described modes of carrying out the
various embodiments of this invention will be apparent to those skilled
in the art based on the above teachings related to the disclosed
invention. The above embodiments of the invention are merely exemplary
and should not be construed to be in any way limiting.